Development of a new translation system for animal proteins: the engineering strategy of ribosome function

新型动物蛋白翻译系统的开发：核糖体功能的工程策略

• 批准号: 16310139
• 负责人: UCHIUMI Toshio
• 金额: $ 10.05万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16310139/
• 关键词: ribosome; protein synthesis; GTPase-associated center; L12; IRES; L11; PO; P1; P2; PO; L7; L10

In this project, we analyzed functional structures in the translational machinery and modified them by molecular engineering in order to develop a new translation system useful for synthesis of eukaryotic proteins. The following are major results of this project.1. The functional structure of protein complex located in the ribosomal GTPase-associated center.In eukaryotes, a couple of P1-P2 heterodimers bound to the C-terminal regions of PO and formed a pentameric complex. In eubacteria, 2〜3 L12 homodimers (differing with species) bound to the C-terminus of L10. In archaebacteria, 3 L12 homodimers bound to PO and formed a heptameric complex, as revealed by biochemical analysis and X-ray crystallography. By removing one or two dimers from each the complex, the activity of GTPase and polypeptide synthesis was reduced.2. Mechanism of translation initiation mediated with the IRES of an insect virus (PSIV).We elucidated that 1) the IRES binds to the ribosome through interaction of the small subunit protein S25, 2) induces some conformational changes in the ribosome, 3) activates the ribosomal accessibility to elongation factor eEF2, and then 4) enhances binding of the first aminoacyl-tRNA to the ribosomal P site. We also succeeded translation of luciferase mediated by the IRES using an IRES-luciferase fusion mRNA. By using this system, we clarified that the 3'-untranlated region of the PSIV IRES-carrying gene participates in the translation efficiency.3. Development of a new in vivo translation system.The E.coli mutant strain dL11 lacks L11 ribosomal protein that is a component of the GTPase-associated center. This strain showed very low growing rate (114〜115 of wild type) and was used for a host cell of protein synthesis. We demonstrated that this E. coli strain is useful for synthesis of eukatyotic proteins that cannot be obtained as a soluble state by usual strains.

在本项目中，我们分析了翻译机制的功能结构，并通过分子工程对其进行修饰，以期开发一种新的翻译系统，用于真核生物蛋白质的合成。以下是本项目的主要成果。蛋白质复合物的功能结构位于核糖体gtpase相关中心。在真核生物中，一对P1-P2异二聚体结合到PO的c端区域并形成一个五聚体复合物。在真细菌中，2 ~ 3个L12同二聚体（不同种类）与L10的c端结合。生化分析和x射线晶体学显示，在古细菌中，3个L12同型二聚体与PO结合形成七聚体配合物。通过从每个复合物中去除一个或两个二聚体，降低了GTPase的活性和多肽的合成。昆虫病毒IRES介导的翻译起始机制我们阐明了1)IRES通过与小亚基蛋白S25的相互作用与核糖体结合，2)诱导核糖体的一些构象变化，3)激活核糖体对延伸因子eEF2的可及性，4)增强第一氨基酰基trna与核糖体P位点的结合。我们还利用IRES-荧光素酶融合mRNA成功翻译了IRES介导的荧光素酶。通过该系统，我们明确了PSIV ires携带基因的3'-非翻译区参与了翻译效率。一种新的体内翻译系统的开发。大肠杆菌突变株dL11缺乏L11核糖体蛋白，该蛋白是gtpase相关中心的一个组成部分。该菌株生长速率极低（野生型114 ~ 115），可作为蛋白质合成的寄主细胞。我们证明了这种大肠杆菌菌株对真核生物蛋白的合成是有用的，而真核生物蛋白是通常菌株无法以可溶性状态获得的。